Zhao, Shuang G.

TPS277 Background: Recent advances in genomic sequencing have deepened our understanding of the molecular complexity of metastatic castration-resistant prostate cancer (mCRPC), revealing multiple actionable alterations that now inform biomarker-driven treatment strategies. Accordingly, germline and somatic tumor profiling is recommended for all patients with mCRPC. Beyond DNA aberrations, gene expression profiling can uncover additional therapeutic targets and pathway activations. The phase 2 PREDICT trial is leveraging both DNA and RNA alterations to guide rationally designed, biomarker-based therapeutic approaches for patients with mCRPC. Methods: This multi-center, multi-arm, biomarker-driven phase 2 umbrella trial is evaluating rationally selected therapies for patients with metastatic castration-resistant prostate cancer (mCRPC). The primary endpoint is objective response rate in patients with measurable disease, with secondary endpoints including radiographic progression-free survival, PSA response, time to first symptomatic skeletal event, overall survival, safety, and correlative biomarker analyses. Eligible patients must have progressive mCRPC of any histology, prior treatment with an androgen receptor pathway inhibitor (ARPI), and either prior or declined taxane chemotherapy, with both measurable and non-measurable disease permitted. Enrollment requires standard-of-care next-generation DNA sequencing from tissue or circulating tumor DNA performed in a CLIA-certified laboratory, and RNA-based allocation will use the CLIA-certified Caris MI Tumor Seek assay (whole exome and whole transcriptome sequencing) on tissue obtained within 12 months of study entry, with a real-time molecular tumor board reviewing results to confirm arm assignment. Patients with RB1 loss (DNA), an RB functional loss signature (RNA), or an NEPC signature (RNA) will receive the EZH1/2 inhibitor valemetostat; those with ≥2 of 3 tumor suppressor gene alterations (TP53, RB1, PTEN), FANC alterations (DNA), or SLFN11 overexpression (RNA) will receive cabazitaxel plus carboplatin; and those without study-defined alterations will receive physician’s choice of cabazitaxel, ARPI, or 177Lu-PSMA-617. The design permits incorporation of additional biomarker-defined arms, and up to 158 patients per arm (64 with measurable and 94 with non-measurable disease) will be accrued under a Simon two-stage minimax design, providing a one-sided type I error of 0.05 if the true response rate is 0.20 and 90% power if the response rate is 0.37 (Clinical trial information: NCT06632977). Clinical trial information: NCT06632977 .

Abstract OBJECTIVE: We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS: Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS: Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION: The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome cancer resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues. Citation Format: Weihua Zhou, Zitong Zhao, Angelica Lin, John Yang, Jie Xu, Kari Wilder-Romans, Annabel Yang, Andrew J. Scott, Jing Li, Sumeet Solanki, Jennifer Speth, Natalie Walker, Ayesha U. Kothari, Yangyang Yao, Erik R. Peterson, Navyateja Korimerla, Christian K. Werner, Jessica Liang, Janna Jacobson, Sravya Palavalasa, Alexandra M Obrien, Ameer L Elaimy, Sean P. Ferris, Shuang G. Zhao, Jann N. Sarkaria, Balázs Győrffy, Shuqun Zhang, Wajd N. Al-Holou, Yoshie Umemura, Meredith A Morgan, Theodore S. Lawrence, Costas A. Lyssiotis, Marc Peters-Golden, Yatrik M. Shah, Daniel R. Wahl. GTP signaling links metabolism, DNA repair, and responses to genotoxic stress [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A004.

Abstract OBJECTIVE We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome GBM resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues.

ABSTRACT How cell metabolism regulates DNA repair is incompletely understood. Here, we define a GTP-mediated signaling cascade that links metabolism to DNA repair and has significant therapeutic implications. GTP, but not other nucleotides, regulates the activity of Rac1, a guanine nucleotide-binding protein, which promotes the dephosphorylation of serine 323 on Abl-interactor 1 (Abi-1) by protein phosphatase 5 (PP5). Dephosphorylated Abi-1, a protein previously not known to activate DNA repair, promotes nonhomologous end joining. In patients and mouse models of glioblastoma, Rac1 and dephosphorylated Abi-1 mediate DNA repair and resistance to standard-of-care genotoxic treatments. The GTP–Rac1–PP5–Abi-1 signaling axis is not limited to brain cancer, as GTP supplementation promotes DNA repair and Abi-1-S323 dephosphorylation in nonmalignant cells and protects mouse tissues from genotoxic insult. This unexpected ability of GTP to regulate DNA repair independently of deoxynucleotide pools has important implications for normal physiology and cancer treatment. SIGNIFICANCE: A newly described GTP-dependent signaling axis is an unexpected link between nucleotide metabolism and DNA repair. Disrupting this pathway can overcome cancer resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues. This article is featured in Selected Articles from This Issue, p. 5

Abstract Introduction/Objectives: Purines regulate DNA repair in brain tumors through uncertain mechanisms. We sought to define the mechanisms by which purines regulate DNA repair and therapy response. Methods: Crispr/Cas9, siRNA, and CDNA overexpression and immunoblot were used to modulate and confirm protein levels. γ-H2AX and Rad51 foci were enumerated using immunofluorescence. Fluorescent reporter assays were performed to detect DNA repair activity. Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after RT and antibodies generated against novel sites. Celltiter-glo or clonogenic assay were performed to evaluate drug/RT responses. IHC was used to detect protein expression. Animal models of glioblastoma and normal tissue (irradiation of GI system) were used to assess DNA repair and treatment responses in vivo. Results: Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT, slowed the repair of RT-induced DSBs and inhibited the activity of non-homologous end joining (NHEJ), but not homologous recombination (HR). These effects could be rescued by GTP (but not ATP) supplementation. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced DSB repair and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to the small GTP-activated protein Rac1. Expression of constitutively active Rac1 promoted the dephosphorylation of Abi-1 (S323) and DSB repair while these effects were blocked by dominant negative Rac1. Pharmacogenomic inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. These findings have therapeutic relevance. In GBM PDX samples, levels of p-Abi-1 (S323) negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibition of Rac1 enhanced RT responses and suppressed Abi-1 S323 dephosphorylation. Abi-1 knockout enhanced efficacy of numerous genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in normal astrocytes and enteroids and protected mice from RT-mediated gastrointestinal injury. Conclusions: GTP promotes DNA repair by activating Rac1 and PP5 to dephosphorylate Abi-1 and stimulate NHEJ. Disrupting this regulation could improve brain tumor treatment responses while augmenting it could help protect normal tissues from genotoxic injury. Citation Format: Weihua Zhou, Angelica Lin, Zitong Zhao, John Yang, Kari Wilder-Romans, Jie Xu, Sumeet Solanki, Jing Li, Annabel Yang, Andrew Scott, Ayesha Kothari, Erik Peterson, Navyateja Korimerla, Jessica Liang, Janna Jacobson, Sravya Palavalasa, Alexandra Obrien, Sean Ferris, Shuang Zhao, Jann N. Sarkaria, Meredith Morgan, Theodore S. Lawrence, Costas A. Lyssiotis, Yatrik Shah, Daniel R. Wahl. GTP-dependent signaling links metabolism, non-homologous end joining and treatment responses in cancer and normal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3498.

Abstract Purpose: Men with metastatic castration-resistant prostate cancer (mCRPC) frequently develop resistance to androgen receptor signaling inhibitor (ARSI) treatment; therefore, new therapies are needed. Trophoblastic cell-surface antigen (TROP-2) is a transmembrane protein identified in prostate cancer and overexpressed in multiple malignancies. TROP-2 is a therapeutic target for antibody–drug conjugates (ADC). Experimental Design: TROP-2 gene (TACSTD2) expression and markers of treatment resistance from prostate biopsies were analyzed using data from four previously curated cohorts of mCRPC (n = 634) and the PROMOTE study (dbGaP accession phs001141.v1.p1, n = 88). EPCAM or TROP-2–positive circulating tumor cells (CTC) were captured from peripheral blood for comparison of protein (n = 15) and gene expression signatures of treatment resistance (n = 40). We assessed the efficacy of TROP-2–targeting agents in a mouse xenograft model generated from prostate cancer cell lines. Results: We demonstrated that TACSTD2 is expressed in mCRPC from luminal and basal tumors but at lower levels in patients with neuroendocrine prostate cancer. Patients previously treated with ARSI showed no significant difference in TACSTD2 expression, whereas patients with detectable AR-V7 expression showed increased expression. We observed that TROP-2 can serve as a cell surface target for isolating CTCs, which may serve as a predictive biomarker for ADCs. We also demonstrated that prostate cancer cell line xenografts can be targeted specifically by labeled anti–TROP-2 agents in vivo. Conclusions: These results support further studies on TROP-2 as a therapeutic and diagnostic target for mCRPC.

295 Background: While 18F-fluciclovine PET/CT is approved in the US and recommended by the NCCN, prostate-specific membrane antigen (PSMA) PET/CT is more common in Europe/Australia and recommended by the EAU. Less is known about the biology of lesions detected by either modality. 18F-fluciclovine PET relies on radiotracer uptake by amino acid transporters LAT1-4 and ASCT1-2. PSMA PET is dependent on surface expression of PSMA. We compared relative expression of PSMA and fluciclovine transporter genes in radical prostatectomy (RP) samples to determine their distribution across subtypes and correlation with outcomes. Methods: Gene expression data of 19,102 RP samples were analyzed using the Affymetrix Human Exon 1.0 ST microarray. 1,135 patients had long term follow up. Associations between expression of PSMA and fluciclovine transporter genes (LAT1-4 and ASCT1-2) and pathologic variables, molecular subtypes, and clinical outcomes were conducted. Results: All fluciclovine transporter genes (LAT 1-4, ASCT1-2) were expressed at lower levels than PSMA (p <0.0001). PSMA expression was positively correlated with genomic risk score and pathologic Gleason score (p<0.0001), but LAT2-3 and ASCT2 were inversely correlated with genomic risk in primary tumors (p<0.0001) and less expressed in GS 9-10 tumors (p<0.0001). PSMA expression was associated with worse metastasis-free survival (MFS) (HR 1.45, p=0.001) and lymph node involvement (HR 2.14, p<0.0001). Expression of LAT2, LAT3, ASCT2 expression was associated with better MFS (HR 0.85, 0.63, 0.74, p<0.0001-0.04). After multivariable adjustment, PSMA expression remained independently prognostic of poorer MFS (HR 1.3, p=0.028). Luminal B subtype was notable for PSMA overexpression; Luminal A was enriched in ASCT2 and LAT2 (p<0.0001). PSMA expression did not correlate with ERG fusion prostate cancers, but LAT2, ASCT1, and ASCT2 were overexpressed in ERG fusion negative tumors (p<0.0001). Conclusions: PSMA expression is associated with more aggressive disease and poorer clinical outcomes than fluciclovine transporter genes in localized prostate cancer. Molecular subtypes of prostate cancer vary in PSMA and fluciclovine transporter gene expression.

BackgroundThe progression of prostate cancer is a complex, multistep process that involves molecular alterations in cells of the tumor and the microenvironment, with associated interactions between the stroma and epithelium. Genomic expression analyses of stromal infiltration markers were performed to determine the significance thereof in prostate cancer.MethodsGenome‐wide expression profiles of formalin‐fixed, paraffin‐embedded radical prostatectomy samples were evaluated from a prospective registry cohort (n = 5239) and 3 retrospective institutional cohorts (n = 1135). Two independent stromal gene expression signatures implied stromal infiltration. Cox proportional hazards regression defined the association between stromal infiltration expression and metastasis‐free survival (MFS).ResultsStromal expression scores were correlated with stromal signature genes and with other key stromal markers (CAV1, VIM, and TAGLN), basal activity, and CD3 and CD4 immune biomarkers (r > 0.5 for all). The top decile of stromal expression was associated with high genomic risk scores (Decipher ≥ 0.6) , high Cancer of the Prostate Risk Assessment–Postsurgical scores, Gleason 9 to 10 disease, and a higher risk for metastasis (hazard ratio, 2.35; 95% CI, 1.37‐4.02; P = .001). A higher stromal infiltration score was also associated with decreased expression of DNA repair genes and higher radiation sensitivity genomic scores. Postoperative radiation therapy (RT) was associated with an MFS benefit for patients with high stromal scores, but not for patients with low stromal scores (Pinteraction = .02).ConclusionsExpression of stromal infiltration markers is correlated with prostate cancer aggressiveness/progression and may be predictive of a response to RT. Stromal infiltration markers should be studied and considered for incorporation into clinical prognostication and decision making.